Mammalian pro-apoptotic Bok genes and their uses

ABSTRACT

Nucleic acid compositions encoding a pro-apoptotic protein, Bok (Bcl-2-related ovarian killer) are identified. Bok has conserved Bcl-2 homology domains 1, 2 and 3 and a C-terminal transmembrane region present in other Bcl-2 related proteins, but lacks the BH4 domain found only in anti-apoptotic Bcl-2 proteins. Over-expression of Bok induces apoptosis. Cell killing induced by Bok is suppressed by co-expression with selective anti-apoptotic Bcl-2 proteins. Bok is highly expressed in the ovary, testis and uterus, particularly in granulosa cells, the cell type that undergoes apoptosis during follicle atresia. Identification of Bok as a new pro-apoptotic protein with wide tissue distribution and hetero-dimerization properties facilitates elucidation of apoptosis mechanisms in reproductive and other tissues, and provides a means for manipulating apoptosis.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Continuation of U.S. application Ser. No.09/517,358 filed Mar. 2, 2000, which is a Divisional of U.S. applicationSer. No. 09/186,250 filed Nov. 4, 1998, now U.S. Pat. No. 6,043,055issued on Mar. 28, 2000, which claims benefit of U.S. ProvisionalApplication Serial No. 60,064,943 filed Nov. 7, 1997.

FEDERAL RESEARCH STATEMENT

[0002] [Federal Research Statement Paragraph] This invention was madewith Government support under Grant no. HDawarded by the NationalInstitutes of Health. The Government may have certain rights in thisinvention.

BACKGROUND OF INVENTION

[0003] Apoptosis or programmed cell death is important during embryonicdevelopment, metamorphosis, tissue renewal, hormone-induced tissueatrophy and many pathological conditions. In multi-cellular organisms,apoptosis ensures the elimination of superfluous cells including thosethat are generated in excess, have already completed their specificfunctions or are harmful to the whole organism. In reproductive tissuesthat are characterized by cyclic functional changes, massive cell deathoccurs under the control of hormonal signals. A growing body of evidencesuggests that the intracellular “death program” activated duringapoptosis is similar in different cell types and conserved duringevolution.

[0004] Apoptosis involves two essential steps. The Bcl-2 family ofproteins that consists of different anti- and pro-apoptotic members isimportant in the “decision” step of apoptosis. In contrast, the“execution” phase of apoptosis is mediated by the activation ofcaspases, cysteine proteases homologous to the C. elegans proteaseced-3, that induce cell death via the proteolytic cleavage of substratesvital for cellular homeostasis. Bcl-2-related proteins act upstream fromcaspases in the cell death pathway and recent studies demonstrated thatanother C. elegans gene, ced-4, or its mammalian homolog Apaf-1 canbridge between Bcl-2/ced-9 family members and caspases.

[0005] The proto-oncogene Bcl-2 was originally isolated at thebreakpoint of the t (14, 18) chromosomal translocation associated withfollicular B-cell lymphoma. Over-expression of Bcl-2 suppressesapoptosis induced by a variety of agents both in vitro and in vivo.Subsequent studies identified a family of Bcl-2-related proteinspossessing several conserved BH (Bcl-2 homology) domains important forhomo- or hetero-dimerization between family members. In addition, aC-terminal transmembrane region for membrane anchoring is also conservedin most members. Based on their differential roles in regulatingapoptosis, the Bcl-2-related proteins can be separated into anti(Bcl-2,Bcl-xL, Mcl-1, Bcl-w and Bfl-1 /A1) and pro-apoptotic members (Bax, BAD,Bak, Bik, Hrk and BID). Through hetero-dimerization, the balance betweenpro- and anti-apoptotic proteins presumably determines cell fate. Theanti-apoptotic effect of Bcl-2 is not universal, however, because Bcl-2over-expression is not effective in blocking Fas-mediated apoptosis andthe apoptosis of auto-reactive thymocytes during negative selection.Recent identification of multiple Bcl-2-related proteins suggests thatselective Bcl-2 members may act in a tissue- and dimerization-specificmanner.

[0006] References

[0007] Bcl related genes are discussed in Yin et al. (1994) Nature369:321-323; Chittenden et al. (1995) EMBO J. 14:5589-5596; and White(1996) Genes Dev. 10:1 Sequences of exemplary bcl-related genes may beaccessed in Genbank. The human hrk gene has the accession no. U76376 andis described in Inohara et al. (1997) EMBO J. 16:1686-1694. The humanbcl-w gene has the accession no. U59747 and is described in Gibson etal. (1996) Oncogene 13 :665-675. Human A1 gene has the accession no.U29680, and is described in Karsan et al. (1996) Blood 87:3089-3096. Thehuman Bak gene has the accession no. U23765, and is described inChittenden et al. (1995) Nature 374:733-736. The human Bak-2 gene hasthe accession no. U16812, and is described in Kiefer et al. (1995)Nature 374 :736-739. The human Bik gene has the accession no. U34584,and is described in Boyd et al. (1995) Oncogene 11 :1921-1928. The humanBfl-1 gene has the accession no. U27467, and is described in Choi et al.(1995) Oncogene 11 :1693-1698. The human bcl-2 gene has the accessionno. M13995, and is described in Tsujimoto and Croce (1986) P.N.A.S. 83:5214-5218. The human Bax genes have the accession nos. L22475, L22474and L22473, and are described in Oltvai et al. (1993) Cell 74 :609-619.The EBV BHRF1 gene has the accession no. A22899, and is described in WOThe human mcl-1 gene is described in Kozopas et al. (1993) P.N.A.S.90:3516-3520, and OMIM 159552.

[0008] The EST fragment, Genbank accession no. AA103989, containspartial sequence of the 5′ end of the mouse Bok gene.

SUMMARY OF INVENTION

[0009] Isolated nucleotide compositions and sequences are provided forBok genes. The provided nucleic acids include splice variants encodinglong forms of the protein, as well as short forms having a truncationthat deletes all or a part of the BH3 domain. The short form of Bok andother related pro-apoptotic proteins may be naturally occurring orsynthetic. These short forms induce cell killing withoutheterodimerization with antiapoptotic proteins.

[0010] The Bok nucleic acid compositions find use in identifyinghomologous or related genes; in producing compositions that modulate theexpression or function of its encoded protein; for gene therapy; mappingfunctional regions of the protein; and in studying associatedphysiological pathways. In addition, modulation of the gene activity inis used for prophylactic and therapeutic purposes, such as treatment ofcancer and other proliferative disorders, identification of cell typebased on expression, and the like.

BRIEF DESCRIPTION OF DRAWINGS

[0011]FIG. 1 is a graph showing quantitative analysis of cell killing byBok and the inhibitory effects of P35. The number of β-gal-expressingcells (mean+/−SEM, n=3) was determined at 36 h after transfection. Datafrom cells transfected with three independent clones (1, 2 and 3)encoding Bok are presented. CHO cells were transfected with a total of2.1 μg plasmid DNA including 2.0 μg of pcDNA3 expression constructs and0.1 μg of the pCMV-β-gal reporter. In cells transfected with twodifferent pcDNA3 expression plasmids, 1.0 μg each was used. Similarresults were obtained in three separate experiments.

[0012]FIG. 2 is a graph showing suppression of Bok-induced apoptosis byselective anti-apoptotic Bcl-2 members in CHO cells. Cell killing by Bokand the antagonistic effects of Mcl-1 and BHRF1 were analyzed. Celltransfection and estimation of apoptosis were as described for FIG. 1.Co-expression of Bcl-2 was ineffective in suppressing Bok-inducedapoptosis.

[0013]FIG. 3 is a schematic representation of wild type Bax and Baktogether with Bax-S and Bak-S constructs with BH3-BH1 deletions similarto that found in Bok-S. The BH domains are boxed and the junctionalsequences derived from the fusion of BH3 and BH1 domains (BH3/1) in themutants are also shown. Numbering of amino acid in the BH1, BH3 andBH3/1 domains are indicated at the bottom of amino acid residues.

DETAILED DESCRIPTION

[0014] Nucleic acid compositions encoding Bok, a pro-apoptotic member ofthe bcl-2 protein family, are provided. Included are splice variantsencoding long forms of the protein, as well as short forms having atruncation that deletes all or a part of the BH3 domain. Also providedare truncated forms of pro-apoptotic proteins related to Bok, e.g. Bax,Bak, etc. These short forms may be naturally occurring or synthetic. Thelong forms associate with anti-apoptotic proteins to form heterodimers,while the short forms induce cell killing without suchheterodimerization.

[0015] As used herein, the term “Bok” is intended to generically referto the polypeptide or nucleic acids as set forth in the Seqlist attachedherewith, homologs thereof, and sequences having substantial similarityand function. Bok occurs naturally in a long form (herein Bok-L), asexemplified by the amino acid sequences provided in SEQ ID NO:2 and SEQID NO:6, which are rat and human, respectively. A short form (hereinBok-S) also occurs naturally, as exemplified by SEQ ID NO:4 and SEQ IDNO:8, in which there is a deletion, leading to the fusion of theN-terminal half of the BH3 domain to the C-terminal half of the BH1domain (herein, BOK-BH3^(inactive)).

[0016] The term “BH3^(inactive)”, or “BH3^(i)” is intended togenerically refer to naturally occurring splice variants and syntheticvariants of Bok or pro-apoptotic Bok-related proteins, e.g. Bax, Bak,etc., in which deletions or amino acid substitutions made in the BH3domain substantially inactivate or abrogate the heterodimerizationactivity of the protein. These variants may also be referred to as“channel only” proteins, because they retain the ability to formchannels in the mitochondria that promote apoptosis.

[0017] The BH3^(i) variants will usually have at least less than about50% of the anti-apoptotic protein binding activity of the parent “long”form, more usually less than about 75% of the anti-apoptotic proteinbinding activity, and preferably less than about 95% of anti-apoptoticprotein binding activity. Examples are provided herein of BH3^(i)variants, including but limited to: alanine substitutions at the highlyconserved Bok glycine 75 residue, truncations of Bax and Bak in the BH3domain, splice variants of Bok where there is a deletion of the aminoacids 76-118; and a glycine substitution was made for leucine 71 toleucine 74 (BokGGGG: 71 LLRL 74 to 71 GGGG 74).

[0018] The BH3 domain has the consensus motif sequence: [SEQ ID NO:11}LRRAGDEFE.RYRR, and generally corresponds to the region of amino acids71-82 in Bok (SEQ ID NO:9 and SEQ ID NO:10). A substitution at theconserved gly75 residue is shown to be sufficient for inactivation.

[0019] Modulation of pro-apoptotic gene activity, which may include Bokor other pro-apoptotic BH3^(i) variants, in vivo is used forprophylactic and therapeutic purposes where it is desirable induce celldeath in specific populations. The specificity of Bok for reproductivetissues is particularly useful in this respect. Diseases where there ishyperproliferation of reproductive tissue, e.g. uterine, testicular andovarian carcinomas, endometriosis, squamous and glandular epithelialcarcinomas of the cervix, etc. are reduced in cell number byupregulating Bok expression to cause apoptosis in susceptible cells.Expression can be regulated by introduction of exogenous Bok genes, orby inducing expression of the native gene. Introduction of exogenous Bokgene and its channel domain only variants into tumor cells followingdirect injection or using tumor-specific carriers can serve as effectivetherapies.

[0020] The isolated Bok genes are useful for in vitro, i.e. cell cultureor cell-free assays, investigation of apoptosis pathways, identificationof cell type based on expression, and the like. The protein is useful asan immunogen for producing specific antibodies, in screening forbiologically active agents that act to regulate Bok gene expression, orthat directly mimic, agonize or antagonize Bok protein function.

[0021] Characterization of Bok

[0022] Bok is expressed mainly in mammalian reproductive tissues,including ovary, testis and uterus. Bok is also expressed in diverseother tissues, albeit at lower levels. It forms a heterodimer withspecific anti-apoptotic Bcl-2 proteins, including mcl-1, BHRF1 andBfl-1. The protein-protein interaction is mediated by the conserved BH1,2 and 3 domain regions of Bok, particularly by the BH3 domain.Overexpression of Bok induces apoptosis in certain cells, particularlyreproductive cells. The rat cDNA sequence is provided as SEQ ID NO:1,the encoded polypeptide product as SEQ ID NO:2. The gene encodes a 213amino acid polypeptide. The rat short form is provided as SEQ ID NO:3,the encoded polypeptide as SEQ ID NO:4. The nucleotide sequences of thehuman long and short forms are provided as SEQ ID NO:5 and 7; theencoded polypeptides as SEQ ID NO:6 and 8.

[0023] Many members of the bcl-2 gene family have been identified andcharacterized, as previously indicated. Other proteins in the pathwayhave also been identified, including caspases, and Apaf-1. Theavailability of isolated genes and gene products in this pathway allowsthe in reconstruction of the pathway and its regulation, using native orgenetically altered molecules, or a combination thereof. Also ofinterest is the use of the genomic region 5′ to Bok or related genes,particularly those members that are hormonally regulated, in order toinvestigate the role of particular transcription factors in regulatingexpression.

[0024] Identification of Bok Sequences

[0025] Homologs of Bok are identified by any of a number of methods. Afragment of the provided cDNA may be used as a hybridization probeagainst a cDNA library from the target organism of interest, where lowstringency conditions are used. The probe may be a large fragment, orone or more short degenerate primers.

[0026] Nucleic acids having sequence similarity are detected byhybridization under low stringency conditions, for example, at 50° C.and 10×SSC (0.9 M NaCl/0.09 M sodium citrate) and remain bound whensubjected to washing at 55° C. in 1×SSC. Sequence identity may bedetermined by hybridization under stringent conditions, for example, at50° C. or higher and 0.1×SSC (9 mM NaCI/0.9 mM sodium citrate). Nucleicacids that are substantially identical to the provided Bok sequences,e.g. allelic variants, genetically altered versions of the gene, etc.,bind to the provided Bok sequences under stringent hybridizationconditions. By using probes, particularly labeled probes of DNAsequences, one can isolate homologous or related genes. The source ofhomologous genes may be any species, e.g. primate species, particularlyhuman; rodents, such as rats and mice, canines, felines, bovines,ovines, equines, yeast, nematodes, etc. Between mammalian species, e.g.human and mouse, homologs have substantial sequence similarity, i.e. atleast 75% sequence identity between nucleotide sequences. Sequencesimilarity is calculated based on a reference sequence, which may be asubset of a larger sequence, such as a conserved motif, coding region,flanking region, etc. A reference sequence will usually be at leastabout 18 nt long, more usually at least about 30 nt long, and may extendto the complete sequence that is being compared. Algorithms for sequenceanalysis are known in the art, such as BLAST, described in Altschul etal. (1990) J Mol Biol 215 :403-10. The sequences provided herein areessential for recognizing Bok related and homologous proteins indatabase searches.

[0027] Bok Nucleic Acid Compositions

[0028] Nucleic acids encoding Bok may be cDNA or genomic DNA or afragment thereof. The term “Bok gene” shall be intended to mean the openreading frame encoding specific Bok polypeptides, e.g. splice variants;introns; as well as adjacent 5″ and 3″ non-coding nucleotide sequencesinvolved in the regulation of expression, up to about 20 kb beyond thecoding region, but possibly further in either direction. The gene may beintroduced into an appropriate vector for extrachromosomal maintenanceor for integration into a host genome.

[0029] The term “cDNA” as used herein is intended to include all nucleicacids that share the arrangement of sequence elements found in nativemature mRNA species, where sequence elements are exons and 3″ and 5″non-coding regions. Normally mRNA species have contiguous exons, withthe intervening introns, when present, removed by nuclear RNA splicing,to create a continuous open reading frame encoding a Bok protein.

[0030] A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It may further include the 3″ and 5″untranslated regions found in the mature mRNA. It may further includespecific transcriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5″ or 3″ end of the transcribedregion. The genomic DNA may be isolated as a fragment of 100 kbp orsmaller; and substantially free of flanking chromosomal sequence. Thegenomic DNA flanking the coding region, either 3′ or 5′, or internalregulatory sequences as sometimes found in introns, contains sequencesrequired for proper tissue and stage specific expression.

[0031] The sequence of the 5′ flanking region may be utilized forpromoter elements, including enhancer binding sites, that provide fordevelopmental regulation in tissues where Bok is expressed. The tissuespecific expression is useful for determining the pattern of expression,for providing promoters that mimic the native pattern of expression, andfor determination of transcription factors that regulate expression.Naturally occurring polymorphisms in the promoter region are useful fordetermining natural variations in expression, particularly those thatmay be associated with disease.

[0032] Alternatively, mutations may be introduced into the promoterregion to determine the effect of altering expression in experimentallydefined systems. Methods for the identification of specific DNA motifsinvolved in the binding of transcriptional factors are known in the art,e.g. sequence similarity to known binding motifs, gel retardationstudies, etc. For examples, see Blackwell et al. (1995) Mol Med 1: 194Mortlock et al. (1996) Genome Res. 6: 327 and Joulin and Richard (1995)Eur J Biochem 232: 620 The regulatory sequences may be used to identifycis acting sequences required for transcriptional or translationalregulation of Bok expression, especially in different tissues or stagesof development, and to identify cis acting sequences and transactingfactors that regulate or mediate Bok expression. Such transcription ortranslational control regions may be operably linked to a Bok gene inorder to promote expression of wild type or altered Bok or otherproteins of interest in cultured cells, or in embryonic, fetal or adulttissues, and for gene therapy. Expression of Bok may be regulatedthrough hormonal control.

[0033] The nucleic acid compositions of the subject invention may encodeall or a part of the subject polypeptides. Double or single strandedfragments may be obtained of the DNA sequence by chemically synthesizingoligonucleotides in accordance with conventional methods, by restrictionenzyme digestion, by PCR amplification, etc. For the most part, DNAfragments will be of at least 15 usually at least 18 or 25 nt, and maybe at least about 50 nt. Such small DNA fragments are useful as primersfor PCR, hybridization screening probes, etc. Larger DNA fragments, i.e.greater than 100 nt are useful for production of the encodedpolypeptide. For use in amplification reactions, such as PCR, a pair ofprimers will be used. The exact composition of the primer sequences isnot critical to the invention, but for most applications the primerswill hybridize to the subject sequence under stringent conditions, asknown in the art. It is preferable to choose a pair of primers that willgenerate an amplification product of at least about 50 nt, preferably atleast about 100 Algorithms for the selection of primer sequences aregenerally known, and are available in commercial software packages.Amplification primers hybridize to complementary strands of DNA, andwill prime towards each other.

[0034] The Bok genes are isolated and obtained in substantial purity,generally as other than an intact chromosome. Usually, the DNA will beobtained substantially free of other nucleic acid sequences that do notinclude a Bok sequence or fragment thereof, generally being at leastabout 50%, usually at least about 90% pure and are typically“recombinant”, i.e. flanked by one or more nucleotides with which it isnot normally associated on a naturally occurring chromosome.

[0035] The DNA may also be used to identify expression of the gene in abiological specimen. The manner in which one probes cells for thepresence of particular nucleotide sequences, as genomic DNA or RNA, iswell established in the literature and does not require elaborationhere. DNA or mRNA is isolated from a cell sample. The mRNA may beamplified by RT using reverse transcriptase to form a complementary DNAstrand, followed by polymerase chain reaction amplification usingprimers specific for the subject DNA sequences. Alternatively, the mRNAsample is separated by gel electrophoresis, transferred to a suitablesupport, e.g. nitrocellulose, nylon, etc., and then probed with afragment of the subject DNA as a probe. Other techniques, such asoligonucleotide ligation assays, in situ hybridizations, andhybridization to DNA probes arrayed on a solid chip may also find use.Detection of mRNA hybridizing to the subject sequence is indicative ofBok gene expression in the sample.

[0036] The sequence of a Bok gene, including flanking promoter regionsand coding regions, may be mutated in various ways known in the art togenerate targeted changes in promoter strength, sequence of the encodedprotein, etc. The DNA sequence or protein product of such a mutationwill usually be substantially similar to the sequences provided herein,i.e. will differ by at least one nucleotide or amino acid, respectively,and may differ by at least two but not more than about ten nucleotidesor amino acids. The sequence changes may be substitutions, insertions ordeletions. Deletions may further include larger changes, such asdeletions of a domain or exon. Of particular interest is the creation ofBH3^(i) variants. Other modifications of interest include epitopetagging, e.g. with the FLAG system, HA, etc. For studies of subcellularlocalization, fusion proteins with green fluorescent proteins (GFP) maybe used.

[0037] Techniques for in vitro mutagenesis of cloned genes are known.Examples of protocols for site specific mutagenesis may be found inGustin et al., Biotechniques 14:22 (1993); Barany, Gene 37:111-23 (1985); Colicelli et al., Mol Gen Genet 199:537-9 (1985); and Prentki etal., Gene 29:303-13 (1984). Methods for site specific mutagenesis can befound in Sambrook et al., Molecular Cloning: A Laboratory Manual, CSHPress 1989, pp. 15.3-15.108; Weiner et Gene 126:35-41 (1993); Sayers etal., Biotechniques 13:592-6 (1992); Jones and Winistorfer, Biotechniques12:528-30 (1992); Barton et al., Nucleic Acids Res 18:7349-55 (1 990);Marotti and Tomich, Gene Anal Tech 6:67(1989); and Zhu, Anal Biochem177:120-4 (1989). Such mutated genes may be used to studystructure-function relationships of Bok, or to alter properties of theprotein that affect its function or regulation.

[0038] Other nucleic acids of the invention include pro-apoptoticBH3^(i) variants. These may be synthesized by using techniques of invitro mutagenesis and genetic engineering to inactivate the BH3 domainof Bok related genes. The wild-type sequence of these genes are knownand publically available, e.g. in Genbank the human Bak gene has theaccession no. U23765; human Bak-2 gene has the accession no. U16812;human Bik gene has the accession no. U34584; human Bax genes have theaccession nos. L22475, L22474 and L22473. One of skill in the art cangenerate the physical nucleic acid from the database sequence by variousmeans, e.g. synthesis of primers and PCR amplification, screening cDNAlibraries, etc.

[0039] Bok Polypeptides

[0040] The subject nucleic acids may be employed for producing all orportions of Bok polypeptides or BH3^(i) variants of pro-apoptotic Bokrelated polypeptides. For expression, an expression cassette may beemployed. The expression vector will provide a transcriptional andtranslational initiation region, which may be inducible or constitutive,where the coding region is operably linked under the transcriptionalcontrol of the transcriptional initiation region, and a transcriptionaland translational termination region. These control regions may benative to a Bok gene, or may be derived from exogenous sources.

[0041] The peptide may be expressed in prokaryotes or eukaryotes inaccordance with conventional ways, depending upon the purpose forexpression. For large scale production of the protein, a unicellularorganism, such as E. coli, B. subtilis, S. cerevisiae, insect cells incombination with baculovirus vectors, or cells of a higher organism suchas vertebrates, particularly mammals, e.g. COScells, may be used as theexpression host cells. In some situations, it is desirable to expressthe Bok gene in eukaryotic cells, where the Bok protein will benefitfrom native folding and post-translational modifications. Small peptidescan also be synthesized in the laboratory. Peptides that are subsets ofthe complete Bok sequence, e.g. peptides of at least about 4 aminoacids, usually at least about 8 amino acids, more usually at least about16 amino acids, up to and including functional domains, and the completeBok polypeptide, may be used to identify and investigate parts of theprotein important for function, or to raise antibodies directed againstthese regions.

[0042] With the availability of the protein or fragments thereof inlarge amounts, by employing an expression host, the protein may beisolated and purified in accordance with conventional ways. A lysate maybe prepared of the expression host and the lysate purified using HPLC,exclusion chromatography, gel electrophoresis, affinity chromatography,or other purification technique. The purified protein will generally beat least about 80% pure, preferably at least about 90% pure, and may beup to and including 100% pure. Pure is intended to mean free of otherproteins, as well as cellular debris.

[0043] The expressed Bok polypeptides are useful for the production ofantibodies, where short fragments provide for antibodies specific forthe particular polypeptide, and larger fragments or the entire proteinallow for the production of antibodies over the surface of thepolypeptide. Epitopes for immunization may comprise one or more of theconserved BH domains. Antibodies may be raised to the wild-type orvariant forms of Bok. Antibodies may be raised to isolated. peptidescorresponding to these domains, or to the native protein.

[0044] Antibodies are prepared in accordance with conventional ways,where the expressed polypeptide or protein is used as an immunogen, byitself or conjugated to known immunogenic carriers, e.g. KLH, pre-SHBsAg, other viral or eukaryotic proteins, or the like. Variousadjuvants may be employed, with a series of injections, as appropriate.For monoclonal antibodies, after one or more booster injections, thespleen is isolated, the lymphocytes immortalized by cell fusion, andthen screened for high affinity antibody binding. The immortalizedcells, i.e. hybridomas, producing the desired antibodies may then beexpanded. For further description, see Monoclonal Antibodies: ALaboratory Manual, Harlow and Lane eds., Cold Spring HarborLaboratories, Cold Spring Harbor, New York, 1988. If desired, the mRNAencoding the heavy and light chains may be isolated and mutagenized bycloning in E. coli, and the heavy and light chains mixed to furtherenhance the affinity of the antibody. Alternatives to in vivoimmunization as a method of raising antibodies include binding to phage“display” libraries, usually in conjunction with in vitro affinitymaturation.

[0045] Modulation of Gene Expression

[0046] The Bok genes, gene fragments, or the encoded protein or proteinfragments, including BH3^(i) variants of related pro-apoptotic sequence,are useful in gene therapy to treat disorders associated with a deficitin pro-apoptosis proteins, including different states of tumorgenesis.This approach is also useful in treating proliferative conditions ofreproductive cells, such as uterine cell hyperplasia, leiomyoma andtumorigenesis. Expression vectors may be used to introduce the codingsequence into a cell. Such vectors generally have convenient restrictionsites located near the promoter sequence to provide for the insertion ofnucleic acid sequences. Transcription cassettes may be preparedcomprising a transcription initiation region, the target gene orfragment thereof, and a transcriptional termination region. Thetranscription cassettes may be introduced into a variety of vectors,e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like,where the vectors are able to transiently or stably be maintained in thecells, usually for a period of at least about one day, more usually fora period of at least about several days to several weeks.

[0047] The exogenous coding sequence or protein may be introduced intotissues or host cells by any number of routes, including viralinfection, microinjection, or fusion of vesicles. Methods that localizethe agent to the particular targeted tissues are of interest.

[0048] Antisense molecules can be used to down-regulate expression ofBok in cells. The anti-sense reagent may be antisense oligonucleotides(ODN), particularly synthetic ODN having chemical modifications fromnative nucleic acids, or nucleic acid constructs that express suchanti-sense molecules as RNA. The antisense sequence is complementary tothe mRNA of the targeted gene, and inhibits expression of the targetedgene products. Antisense molecules inhibit gene expression throughvarious mechanisms, e.g. by reducing the amount of mRNA available fortranslation, through activation of RNAse H, or steric hindrance. One ora combination of antisense molecules may be administered, where acombination may comprise multiple different sequences.

[0049] Antisense molecules may be produced by expression of all or apart of the target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996) Nature Biotechnology 14 :840-844).

[0050] A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

[0051] Antisense oligonucleotides may be chemically synthesized bymethods known in the art (see Wagner et al. (1993) supra. and Milliganet al., supra.) Preferred oligonucleotides are chemically modified fromthe native phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications have been described in the literature, which alter thechemistry of the backbone, sugars or heterocyclic bases.

[0052] Among useful changes in the backbone chemistry arephosphorothioates; phosphorodithioates, where both of the non-bridgingoxygens are substituted with sulfur; phosphoroamidites; alkylphosphotriesters and boranophosphates. Achiral phosphate derivativesinclude 3″-O″-5″-S-phosphorothioate, 3″-S-5″-O-phosphorothioate,3″-CH2-5″-O-phosphonate and 3″-NH-5″-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The α-anomer of deoxyribose may be used, where the base isinverted with respect to the natural β-anomer. The 2″-OH of the ribosesugar may be altered to form 2″-O-methyl or 2″-O-allyl sugars, whichprovides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5″-deoxycytidine and 5-bromo-2″-deoxycytidine fordeoxycytidine. 5propynyl-2″-deoxyuridine and 5-propynyl-2″-deoxycytidinehave been shown to increase affinity and biological activity whensubstituted for deoxythymidine and deoxycytidine, respectively.

[0053] Genetically Altered Cell or Animal Models for Bok Function

[0054] The subject nucleic acids can be used to generate transgenicanimals or site specific gene modifications in cell lines. Transgenicanimals may be made through homologous recombination, where the normalBok locus is altered. Alternatively, a nucleic acid construct israndomly integrated into the genome. Vectors for stable integrationinclude plasmids, retroviruses and other animal viruses, YACs, and thelike.

[0055] The modified cells or animals are useful in the study ofpro-apoptotic gene function and regulation. For example, a series ofsmall deletions and/or substitutions may be made in the Bok gene todetermine the role of different exons in oncogenesis, signaltransduction, etc. Of interest are the use of Bok to constructtransgenic animal models for proliferative disorders, e.g.endometriosis, where expression of Bok is specifically reduced orabsent. Specific constructs of interest include anti-sense Bok, whichwill block Bok expression, expression of dominant negative Bokmutations. A detectable marker, such as lac Z may be introduced into theBok locus, where upregulation of Bok expression will result in an easilydetected change in phenotype.

[0056] One may also provide for expression of the Bok gene or variantsthereof in cells or tissues where it is not normally expressed or atabnormal times of development. By providing expression of Bok protein incells in which it is not normally produced, one can induce changes incell behavior.

[0057] DNA constructs for homologous recombination will comprise atleast a portion of the Bok gene with the desired genetic modification,and will include regions of homology to the target locus. Conveniently,markers for positive and negative selection are included. Methods forgenerating cells having targeted gene modifications through homologousrecombination are known in the art. For various techniques fortransfecting mammalian cells, see Keown et al. (1990) Methods inEnzymology 185: 527-537.

[0058] For embryonic stem (ES) cells, an ES cell line may be employed,or embryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of leukemia inhibitingfactor (LIF). When ES or embryonic cells have been transformed, they maybe used to produce transgenic animals. After transformation, the cellsare plated onto a feeder layer in an appropriate medium. Cellscontaining the construct may be detected by employing a selectivemedium. After sufficient time for colonies to grow, they are picked andanalyzed for the occurrence of homologous recombination or integrationof the construct. Those colonies that are positive may then be used forembryo manipulation and blastocyst injection. Blastocysts are obtainedfrom 4 to 6 week old superovulated females. The ES cells aretrypsinized, and the modified cells are injected into the blastocoel ofthe blastocyst. After injection, the blastocysts are returned to eachuterine horn of pseudopregnant females. Females are then allowed to goto term and the resulting offspring screened for the construct. Byproviding for a different phenotype of the blastocyst and thegenetically modified cells, chimeric progeny can be readily detected.

[0059] The chimeric animals are screened for the presence of themodified gene and males and females having the modification are mated toproduce homozygous progeny. If the gene alterations cause lethality atsome point in development, tissues or organs can be maintained asallogeneic or congenic grafts or transplants, or in in vitro culture.The transgenic animals may be any non-human mammal, such as laboratoryanimals, domestic animals, etc. The transgenic animals may be used infunctional studies, drug screening, etc.

[0060] In Vitro Models for Bok Function

[0061] The availability of a number of members in the bcl-2 gene family,as previously described, allows in vitro reconstruction of the apoptosispathway. Two or more of the components may be combined in vitro, and thebehavior assessed in terms of activation of transcription of specifictarget sequences; modification of protein components, e.g. proteolyticprocessing, phosphorylation, methylation, etc.; ability of differentprotein components to bind to each other, etc. The components may bemodified by sequence deletion, substitution, etc. to determine thefunctional role of specific domains.

[0062] Drug screening may be performed using an in vitro model, agenetically altered cell or animal, or purified protein. One canidentify ligands or substrates that bind to, modulate or mimic theaction of Bok and other pro-apoptotic proteins. Areas of investigationinclude the development of cancer treatments, agents that modulate Bokexpression, etc. Drug screening identifies agents that provide areplacement for Bok function in abnormal cells. Conversely, agents thatreverse Bok function may stimulate controlled growth and healing. Ofparticular interest are screening assays for agents that have a lowtoxicity for human cells. A wide variety of assays may be used for thispurpose, including labeled in vitro protein-protein binding assays,electrophoretic mobility shift assays, immunoassays for protein binding,and the like. The purified protein may also be used for determination ofthree-dimensional crystal structure, which can be used for modelingintermolecular interactions.

[0063] The term “agent” as used herein describes any molecule, e.g.protein or pharmaceutical, with the capability of altering or mimickingthe physiological function of Bok. Generally a plurality of assaymixtures are run in parallel with different agent concentrations toobtain a differential response to the various concentrations. Typically,one of these concentrations serves as a negative control, i.e. at zeroconcentration or below the level of detection.

[0064] Candidate agents encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 50 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

[0065] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

[0066] Where the screening assay is a binding assay, one or more of themolecules may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

[0067] A variety of other reagents may be included in the screeningassay. These include reagents like salts, neutral proteins, e.g.albumin, detergents, etc that are used to facilitate optimalprotein-protein binding and/or reduce non-specific or backgroundinteractions. Reagents that improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.may be used. The mixture of components are added in any order thatprovides for the requisite binding. Incubations are performed at anysuitable temperature, typically between 4 and 40° C. Incubation periodsare selected for optimum activity, but may also be optimized tofacilitate rapid high-throughput screening. Typically between 0.1 and 1hours will be sufficient.

[0068] Other assays of interest detect agents that mimic Bok function.For example, an expression construct comprising a Bok gene may beintroduced into a cell line under conditions that allow expression. Thelevel of Bok activity is determined by a functional assay. In onescreening assay, candidate agents are added, and the ability todown-regulate its activity is detected. In another assay, the ability ofcandidate agents to enhance Bok function is determined. Alternatively,candidate agents are added to a cell that lacks functional Bok, andscreened for the ability to reproduce Bok in a functional assay.

[0069] The compounds having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a host fortreatment of proliferative diseases, etc. The compounds may also be usedto enhance Bok function. The inhibitory agents may be administered in avariety of ways, orally, topically, parenterally e.g. subcutaneously,intraperitoneally, by viral infection, intravascularly, etc. Topicaltreatments are of particular interest. Depending upon the manner ofintroduction, the compounds may be formulated in a variety of ways. Theconcentration of therapeutically active compound in the formulation mayvary from about 0.1 The pharmaceutical compositions can be prepared invarious forms, such as granules, tablets, pills, suppositories,capsules, suspensions, salves, lotions and the like. Pharmaceuticalgrade organic or inorganic carriers and/or diluents suitable for oraland topical use can be used to make up compositions containing thetherapeutically-active compounds. Diluents known to the art includeaqueous media, vegetable and animal oils and fats. Stabilizing agents,wetting and emulsifying agents, salts for varying the osmotic pressureor buffers for securing an adequate pH value, and skin penetrationenhancers can be used as auxiliary agents.

[0070] Diagnostic Uses

[0071] The subject nucleic acid and/or polypeptide compositions may beused to analyze a patient sample for the presence of polymorphismsassociated with a disease state or genetic predisposition to a diseasestate. Biochemical studies may be performed to determine whether asequence polymorphism in a Bok coding region or control regions isassociated with disease. Disease associated polymorphisms may includedeletion or truncation of the gene, mutations that alter expressionlevel, etc. Changes in the promoter or enhancer sequence that may affectexpression levels of Bok can be compared to expression levels of thenormal allele by various methods known in the art. Methods fordetermining promoter or enhancer strength include quantitation of theexpressed natural protein; insertion of the variant control element intoa vector with a reporter gene such as α galactosidase, luciferase,chloramphenicol acetyltransferase, etc. that provides for convenientquantitation; and the like.

[0072] A number of methods are available for analyzing nucleic acids forthe presence of a specific sequence, e.g. a disease associatedpolymorphism. Where large amounts of DNA are available, genomic DNA isused directly. Alternatively, the region of interest is cloned into asuitable vector and grown in sufficient quantity for analysis. Cellsthat express Bok may be used as a source of mRNA, which may be assayeddirectly or reverse transcribed into cDNA for analysis. The nucleic acidmay be amplified by conventional techniques, such as the polymerasechain reaction (PCR), to provide sufficient amounts for analysis. Theuse of the polymerase chain reaction is described in Saiki, et al.(1985) Science 239:487, and a review of techniques may be found inSambrook, et al. Molecular Cloning: A Laboratory Manual, CSH Press 1989,pp.14.214.33. Alternatively, various methods are known in the art thatutilize oligonucleotide ligation as a means of detecting polymorphisms,for examples see Riley et al. (1 990) N.A.R. 18:2887-2890; and Delahuntyet al. (1996) Am. J. Hum. Genet. 58:1239-1246.

[0073] A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM), 2″,7″″,5″-dichloro-6-carboxyfluorescein(JOE), 6-carboxy-X-rhodamine (ROX), 6″,4″,7″,4,7-hexachloroflourescein(HEX), 5(5 or N, N,N″,N″-tetramethyl-6-carboxyrhodamine (TAMRA),radioactive labels, e.g. ³⁵P, ³⁵S, ³H; etc. The label may be a two stagesystem, where the amplified DNA is conjugated to biotin, haptens, etc.having a high affinity binding partner, e.g. avidin, specificantibodies, etc., where the binding partner is conjugated to adetectable label. The label may be conjugated to one or both of theprimers. Alternatively, the pool of nucleotides used in theamplification is labeled, so as to incorporate the label into theamplification product.

[0074] The sample nucleic acid, e.g. amplified or cloned fragment, isanalyzed by one of a number of methods known in the art. The nucleicacid may be sequenced by dideoxy or other methods, and the sequence ofbases compared to a wild-type Bok sequence. Hybridization with thevariant sequence may also be used to determine its presence, by Southernblots, dot blots, etc. The hybridization pattern of a control andvariant sequence to an array of oligonucleotide probes immobilised on asolid support, as described in U.S. Pat. No. 5,445,934, or inWO95/35505, may also be used as a means of detecting the presence ofvariant sequences. Single strand conformational polymorphism (SSCP)analysis, denaturing gradient gel electrophoresis (DGGE), andheteroduplex analysis in gel matrices are used to detect conformationalchanges created by DNA sequence variation as alterations inelectrophoretic mobility. Alternatively, where a polymorphism creates ordestroys a recognition site for a restriction endonuclease, the sampleis digested with that endonuclease, and the products size fractionatedto determine whether the fragment was digested. Fractionation isperformed by gel or capillary electrophoresis, particularly acrylamideor agarose gels.

[0075] Screening for mutations in Bok may be based on the functional orantigenic characteristics of the protein. Protein truncation assays areuseful in detecting deletions that may affect the biological activity ofthe protein. Various immunoassays designed to detect polymorphisms inBok proteins may be used in screening. Where many diverse geneticmutations lead to a particular disease phenotype, functional proteinassays have proven to be effective screening tools. The activity of theencoded Bok protein may be determined by comparison with the wild-typeprotein.

[0076] Antibodies specific for a Bok may be used in staining or inimmunoassays. Samples, as used herein, include biological fluids such assemen, blood, cerebrospinal fluid, tears, saliva, lymph, dialysis fluidand the like; organ or tissue culture derived fluids; and fluidsextracted from physiological tissues. Also included in the term arederivatives and fractions of such fluids. The cells may be dissociated,in the case of solid tissues, or tissue sections may be analyzed.Alternatively a lysate of the cells may be prepared.

[0077] Diagnosis may be performed by a number of methods to determinethe absence or presence or altered amounts of normal or abnormal Bok inpatient cells. For example, detection may utilize staining of cells orhistological sections, performed in accordance with conventionalmethods. Cells are permeabilized to stain cytoplasmic molecules. Theantibodies of interest are added to the cell sample, and incubated for aperiod of time sufficient to allow binding to the epitope, usually atleast about 10 minutes. The antibody may be labeled with radioisotopes,enzymes, fluorescers, chemiluminescers, or other labels for directdetection. Alternatively, a second stage antibody or reagent is used toamplify the signal. Such reagents are well known in the art. Forexample, the primary antibody may be conjugated to biotin, withhorseradish peroxidase-conjugated avidin added as a second stagereagent. Alternatively, the secondary antibody conjugated to aflourescent compound, e.g. flourescein, rhodamine, Texas red, etc. Finaldetection uses a substrate that undergoes a color change in the presenceof the peroxidase. The absence or presence of antibody binding may bedetermined by various methods, including flow cytometry of dissociatedcells, microscopy, radiography, scintillation counting, etc. Diagnosticscreening may also be performed for polymorphisms that are geneticallylinked to a disease predisposition, particularly through the use ofmicrosatellite markers or single nucleotide polymorphisms. Frequentlythe microsatellite polymorphism itself is not phenotypically expressed,but is linked to sequences that result in a disease predisposition.However, in some cases the microsatellite sequence itself may affectgene expression. Microsatellite linkage analysis may be performed alone,or in combination with direct detection of polymorphisms, as describedabove. The use of microsatellite markers for genotyping is welldocumented. For examples, see Mansfield et al. (1994) Genomics24:225-233; Ziegle et al. (1992) Genomics 14:1026-1031; Dib et al.,supra. It is to be understood that this invention is not limited to theparticular methodology, protocols, formulations and reagents described,as such may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0078] It must be noted that as used herein and in the appended claims,the singular forms “a”, “and”, and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a complex” includes a plurality of such complexes and reference to “theformulation” includes reference to one or more formulations andequivalents thereof known to those skilled in the art, and so forth.

[0079] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this invention belongs. Although any methods,devices and materials similar or equivalent to those described hereincan be used in the practice or testing of the invention, the preferredmethods, devices and materials are now described.

[0080] All publications mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, themethods, ligands, and methodologies that are described in thepublications which might be used in connection with the presentlydescribed invention. The publications discussed above and throughout thetext are provided solely for their disclosure prior to the filing dateof the present application. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention.

[0081] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the subject invention, and are not intended to limitthe scope of what is regarded as the invention. Efforts have been madeto ensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight, andpressure is at or near atmospheric.

[0082] Experimental

EXAMPLE 1

[0083] Isolation and Characterization of Bok cDNA

[0084] Materials and Methods

[0085] Two-Hybrid Screening

[0086] The full-length open reading frame (ORF) of rat Mcl-1 cDNA wasfused in frame with the GAL4-binding domain (GAL4-BD) into the pGBT-9yeast shuttle vector (Clontech, Palo Alto, Calif.). This vector was usedto identify Mcl-1-interacting proteins by screening 1.5 milliontransformants from a GAL4-activation domain (AD)-tagged ovarian fusionMatchmaker cDNA library. The ovarian cDNAs were prepared from 27-day-oldSprague-Dawley rats primed for 36 h with 10 IU equine gonadotropin.Yeast cells were first transformed with pGBT9-Mcl-1 and coloniesselected in plates deficient for tryptophan. In the second step, cellswere transformed with cDNAs from the ovarian library before selection ofclones in plates lacking tryptophan, leucine and histidine. Positivetransformants were further selected for growth in media containing 5 mM3-aminotriazole. Individual AD-fusion cDNAs were retrieved followingtransformation of E. coli cells.

[0087] A total of 40 potential Mcl1-interacting clones were re-screenedagainst the empty vector or vector encoding different Bcl-2 proteins toeliminate false positives. Three clones of Bok cDNAs were isolated basedon their ability to interact with Mcl-1 in an HF7c yeast reporter strain(Fields & Song (1 989) Nature 340, 245-247). DNA sequence analysis andcomparison with known genes using the BLASTX algorithm revealed that thepositive clones encode a polypeptide sharing high homology withBclproteins. Further analysis of expressed sequence tags (EST) in theGenBank revealed that EST accession number AA103989 has greater than 98%identity with the 5′-sequence of cloned cDNA and contains extra5′-sequence of the murine Bok homolog. Full-length ORF and5′-untranslated sequence of rat Bok were obtained by PCR using theGAL4-AD-tagged ovarian Matchmaker cDNA library as the template and anupstream primer based on murine EST. Complementary DNA fragments with anidentical ORF were also obtained in separate PCR using a rat brain cDNAlibrary (Stratagene, La Jolla, Calif.) as the template. Interactionsbetween Bok and different Bclmembers were assessed in the yeasttwo-hybrid system using pGBT9 GAL4-BD and pGADGH GAL4-AD vectors (Bartelet al. (1993) in Cellular Interaction in Development: A PracticalApproach, ed. Hartley, D. A. (Oxford University Press, Oxford), pp.153-179). Specific binding of different protein pairs was evaluatedbased on the activation of GAL1-HIS3 and GAL4-lacZ reporter genes.

[0088] Cell Culture and Transfection with Plasmids

[0089] For the expression of Bcl-2 proteins in eukaryotic cells,PCR-generated ORF of different cDNAs were subcloned into the pcDNA3vector (Invitrogen, Inc., San Diego, Calif.). Following transfection ofcDNAs, cell death was monitored (Kumar et al. (1994) Genes Dev. 8,1613-1626). CHO cells (2×10⁵/well) were cultured in Dulbecco's modifiedEagle's medium (DMEM)/F1 2 supplemented with 10% fetal bovine serum, 100U/ml penicillin, 100 μg /ml streptomycin and 2 mM glutamine. One daylater, cells were transfected using the lipofectamine procedure (LifeTechnologies, Gaithersburg, Md.) with the empty pcDNA3 expression vectoror the same vector containing different cDNAs, together with {fraction(1/10)}-{fraction (1/20)} fractions of an indicator plasmid pCMV-β-galto allow the identification of transfected cells. Inclusion of 10- to20-fold excess of expression vectors as compared to the pCMV-β-galreporter plasmid ensured that most of the β-galactosidase expressingcells also expressed the protein(s) under investigation. Cells wereincubated with plasmids in a serum-free medium for 4 h, followed byadding fetal bovine serum to a final concentration of 5% and furtherincubation for 14 h. After an additional culture in fresh medium for 1 8h, cells were fixed by 0.25% glutaraldehyde and stained with X-gal (0.4mg/ml) to detect β-galactosidase expression. The number of blue cellswas counted by microscopic examination (Kumar et al. (1 994) Genes Dev.8, 1613-1626). To verify the nature of cell death, total cellular DNAwas extracted for 3″-end labeling of DNA ends at 18 h after transfectionwith ³²P-ddATP before gel fractionation to identify internucleosomal DNAfragmentation (Hsu et al. (1996) Endocrinology 137, 4837-4843).Statistical differences among treatment groups were analyzed usingone-way ANOVA and Scheffe F-test.

[0090] Northern and Southern Blots and In Situ Analyses

[0091] For Northern blot analysis of Bok expression, tissues werecollected from 27-day-old Sprague-Dawley rats (Simonsen Lab, Gilroy,Calif.). For Bok expression in ovarian cells, ovaries were obtained from26-day-old rats implanted for two days with a diethylstilbestrol capsuleto stimulate development of multiple early antral follicles (Bicsak etal. (1986) Endocrinology 119, 2711-2719). Granulosa cells were preparedby needle puncture. For the extraction of total RNA, tissues werehomogenized in Tri-Reagent solution (Molecular Research Center, Inc.,Cincinnati, Ohio) and at least two pools from each treatment group wereused. In addition, poly (A+) RNA was isolated using the Oligotexoligo-dT resin (Qiagen, Inc., Chatsworth, Calif.). Aliquots of eachsample were denatured and fractionated in 1% agarose gels containingformaldehyde before northern blotting analysis. Membranes werepre-hybridized for 4 h at 65° C. in a solution containing 50% formamide,5×sodium phosphate buffer (SSPE), 5×Denhardt's solution, 0.5% SDS and500 μg/ml yeast tRNA. This was followed by overnight hybridization inthe same conditions but with 1×10⁶ cpm/ml of ³²P-labeled Bok or GAPDHcRNA probe. After hybridization, the membranes were washed twice in2×SSC, 1% SDS at room temperature, followed by two washes in 0.1×SSC, 1%SDS at 65° C. before exposure to Kodak RX films (Eastman Kodak,Rochester, N.Y.). For studies on the conservation of the Bok gene duringevolution, the Zoo blot (Clontech) containing genomic DNA from differentvertebrate species was hybridized with a ³²P-labeled rat Bok cDNA probeunder stringent conditions.

[0092] For in situ hybridization analysis of Bok mRNA expression,ovaries from 26-day-old rats were isolated and fixed at 4° C. for 4 h in4% paraformaldehyde in PBS (pH 7.4), followed by overnight dehydrationin 0.5M sucrose. Tissue blocks were embedded in Tissue-Tek solution(Sakura Finetek USA Inc., Torrence, Calif.) and snapped frozen in liquidnitrogen. Twelve μm thick cryo-sections were mounted on chargedmicroscopic slides (Fischer Scientific, Pittsburgh, Pa.), post-fixed in4% paraformaldehyde and stored at −70° C. for up to 1 month.Hybridization and washes of cryosections were as previously described(Hsu et al., supra. ). After two weeks of exposure under NTB2 emulsion(Kodak), the slides were developed, counter-stained and mounted withPermount (Fisher Scientific, Fair Lawn, N.J.) for observation andphotography using a Nikon Optiphot microscope.

[0093] Results

[0094] Using the anti-apoptotic protein Mcl-1 as bait, we screened anovarian fusion cDNA library and isolated three Bok clones. SubsequentDNA sequencing and identification of homologous murine ESTs allowedisolation of full-length Bok cDNAs following PCR of ovarian and braincDNA libraries. The ORF of Bok encoded a protein of 213 amino acidsshowing no identity with any known gene. The novel protein has apredicted molecular weight of 23.5 kD and a pl of 9.1. The methionineinitiation codon conformed to the consensus Kozak sequence andhydrophobicity analysis predicted the presence of a C-terminaltransmembrane domain. In addition, two potential phosphorylation siteswere found near the N-terminal region. Comparison of DNA sequences amongdifferent Bcl-2 proteins indicated that Bok was a novel member of thisfamily showing conserved BH 1, 2 and 3 domains. However, the BH4 domain,known to be important for the anti-apoptotic function of mammalian Bcl-2proteins, was missing in Bok. Closer comparison indicated the core BH1domain of Bok (TWGK) was less conserved as compared with other Bcl-2proteins (NWGR). For the BH3 domain found in pro-apoptotic members, thecore sequence (GDE) was conserved in Bok but the flanking sequences weredifferent. Furthermore, analysis of the phylogenetic relatedness of thedifferent Bcl-2 members suggests that, during evolution, Bok divergedearly from other Bcl-2 proteins.

[0095] We investigated interactions between Bok and different anti- andpro-apoptotic Bcl-2 proteins. Bok only interacts with selectiveanti-apoptotic Bcl-2 proteins in the yeast two-hybrid system. Yeastcells were grown in the selective media containing 53-aminotriazole andwithout Trp, Leu and His. Prominent growth of yeast colonies expressingBok fused to the GAL4 activation domain together with Mcl-1, BHRF1 orBfl-1 fused to the GAL4 binding domain could be seen. Minimal growth ofyeast colonies was found in cells that express the same Bok expressingvector together with Bcl-2, Bcl-xL, Bcl-w, BAD, Bax, Bak or Bik fused tothe GAL4 binding domain. In addition, prominent growth of coloniesexpressing Bcl-xL and different pro-apoptotic Bcl-2 proteins indicatedthat the lack of growth in yeast cells expressing Bok and differentpro-apoptotic family members was not due to suppression of cell growthby these pro-apoptotic proteins.

[0096] Of interest, Bok did not interact with any pro-apoptotic memberstested. To demonstrate that the lack of interactions between Bok andpro-apoptotic Bcl-2 proteins was not due to the killing of yeast cellsby these apoptosis agonists, we also tested the growth of yeast cellsco-transformed with Bcl-xL and different pro-apoptotic proteins.Although Bcl-xL showed negligible interaction with Bok, it interactedstrongly with all the pro-apoptotic members tested.

[0097] To further study the restricted dimerization property of Bok withselective anti-apoptotic proteins, we tested the growth of yeast cellsthat were co-transformed with different pairs of pro- and anti-apoptoticBcl-2 proteins. Several pro-apoptotic proteins (Bak, Bik and Bax),unlike Bok, all interacted strongly with diverse anti-apoptotic proteinstested, data shown in Table 1, suggesting the restrictedhetero-dimerization property of Bok was unique. [t1] TABLE 1 pGBT-9BHRF1 Mcl-1 Bfl-1 Bcl-2 Bcl-xL Bcl-w Bok −− ++ ++ + −− −− −− Bax −− ++++ ++ ++ ++ ++ Bak −− ++ ++ ++ ++ ++ ++ Bik −− ++ ++ ++ ++ ++ ++

[0098] Summary of protein-protein interactions between pairs ofpro-apoptotic proteins (Bok, Bak, Bik and Bax) and differentanti-apoptotic Bcl-2 members. The positive signs indicate prominent (++)or moderate (+) yeast cell growth whereas the negative signs (−)indicate the absence of reporter gene expression.

[0099] The ability of Bok to regulate apoptosis in mammalian cells wasinvestigated. In CHO cells, transfection with expression vectorsencoding Bok for 36 h induced cell death. The pro-apoptotic effect ofBok was specific because transfection of the empty plasmid or the sameplasmid containing Bok cDNA in reverse orientation did not affect cellsurvival. Furthermore, co-expression of P35, a cysteine proteaseinhibitor derived from the baculovirus (Bump et al. (1995) Science 269,1885-1888), prevented Bok-induced cell killing as indicated by increasesin the number of viable cells.

[0100] Normal cell morphology was found in cells transiently transfectedwith the empty pcDNA3 expression vector (2.1 μg DNA/35 mm dish) or thevector containing Bok cDNA in reverse orientation. Cells were alsotransfected with the Bok expression vector without or with an equalamount of the P35-expressing construct.

[0101] The 3″-end labeling of genomic DNA fragments at an earlier timepoint (18 h) further demonstrated the induction of internucleosomal DNAfragmentation following Bok over-expression, confirming the induction ofapoptosis. The observed DNA fragmentation was blocked by co-expressionwith P35. CHO cells were treated as described in above. At 18 h aftertransfection, cellular DNA was extracted for analysis of DNAfragmentation using a 3″-end labeling method.

[0102] Quantitative analysis also indicated that Bok over-expressiondecreased viable cell number by 75% whereas co-expression of P35completely reversed Bok killing (Fig. substantiating the involvement ofcaspases in Bok action.

[0103] We further tested if the restricted hetero-dimerization of Bokwith selective anti-apoptotic Bcl-2 members found in the yeasttwo-hybrid system could be substantiated in mammalian cells. Bok wasco-expressed with Mcl-1, BHRF1 or Bcl-2 in CHO cells. As shown in FIG.2, Bok-induced apoptosis was attenuated following co-expression withMcl-1 or BHRF1; but co-expression with Bcl-2 was ineffective in blockingBok action. Transfection of the same Bcl-2 expression vector was,however, capable of blocking apoptosis induced by staurosporin.

[0104] The expression of Bok mRNA in diverse rat tissues was examined.High levels of Bok transcript of ˜1.5 kb in size were abundant in theovary, testis and uterus, less abundant in the brain, heart andintestine and negligible in other tissues examined. Further analysis ofBok mRNA in isolated granulosa cells demonstrate high levels ofexpression in these cells that undergo apoptosis during follicledegeneration. In situ hybridization analyses further confirmed highlevels of the Bok transcript in the granulosa cells of antral andpreantral follicles, with minimal signals in theca and interstitialcellsFor northern blot analysis, poly (A)+-selected RNA from differenttissues of rats at 27 days of age or from isolated granulosa cells ofestrogen-treated rats were hybridized with a ³²P-labeled Bok cRNA probe.After washing, the blots were exposed to X-ray films at −70° C. for fivedays. Subsequent hybridization with a GAPDH cRNA probe was performed toestimate nucleic acid loading (8 h exposure). For in situ hybridizationanalysis, ovaries from immature eCG-treated rats were probed with theanti-sense Bok cRNA. Positive signals were found in granulosa cells ofpreantral follicles and an antral follicle. No signal was found in asection hybridized with the sense Bok probe.

[0105] Conservation of the Bok gene in diverse vertebrates was testedusing Southern blot hybridization of genomic DNA from different species.DNA was digested with the EcoRI enzyme and probed with a Bok cDNA probe.Following hybridization at 68° C., the membrane was washed under highstringency conditions (1% SDS, 0.1×SSC at 65° C.) before exposure. Underhigh stringency washing conditions, the rat cDNA cross-hybridizedstrongly with rat, human and monkey genomic DNA and weakly with dog, cowand rabbit DNA. Negligible hybridization signals were found for chickenDNA.

[0106] A cDNA encoding the human bok gene was isolated from a humanovary cDNA library. A partial sequence of the human protein is providedin SEQ ID NO:3.

[0107] Discussion

[0108] A new pro-apoptotic Bcl-2-related protein Bok has been identifiedbased on its binding to an ovarian anti-apoptosis protein Mcl-1. Inaddition to its elevated expression in several reproductive tissues, Bokwas found in diverse other tissues. In addition, Bok shows a selectivehetero-dimerization property by interacting with some (Mcl-1, BHRF1 andBfl-1) but not other (Bcl-2, Bcl-xL and Bcl-w) anti-apoptotic proteins.Coupled with findings that Bok-induced apoptosis could only beantagonized by selective anti-apoptotic proteins, the present datasuggest that different pro- and anti-apoptotic Bcl-2 protein pairs mayplay tissue-specific roles in the regulation of apoptosis. Due to thehigher expression of Bok to ovarian granulosa cells and severalreproductive tissues characterized by hormonally regulated cyclic cellturnover, further analyses of Bok action in the gonads and uterus couldprovide unique models to study the hormonal regulation of apoptosis.Because most of the Bcl-2-related proteins have been identified in thelymphoid system, the present yeast two-hybrid screen provides anexperimental paradigm to isolate novel Bcl-2 homologs essential forapoptosis regulation in other tissues.

[0109] Although the mechanism by which the Bcl-2 proteins participatesin the “decision” step of apoptosis is not clear, the ratio of anti- andpro-apoptotic Bcl-2 members and their hetero- and homo-dimerization arebelieved to determine whether a cell will respond to an apoptoticsignal. Among the Bcl-2 family of proteins, several homology domainshave been found to be essential for their function. Bok containsconserved BH1, 2 and 3 domains but lacks the BH4 domain found in mostanti-apoptotic members. In addition, the conserved NH1 region importantfor the survival function of several anti-apoptotic Bcl-2 proteins isalso absent in Bok. Consistent with its structural features,over-expression of Bok in CHO cells induces apoptosis based on observedcell morphology and internucleosomal DNA fragmentation. Bok-induced cellkilling, like that induced by Bax and BAD, is mediated by caspases asdemonstrated by the suppressive actions of the baculoviral P35 protein.

[0110] Among the pro-apoptotic Bcl-2 proteins, Bok is most similar toBax and Bak in having the BH1, 2 and 3 domains plus the C-terminaltransmembrane sequence. Studies on Bax and Bak with truncation indifferent BH domains suggested that these pro-apoptotic proteins mightexert their effects by hetero-dimerizing with Bcl-2 or Bcl-xL.Competitive dimerization between selective pairs of anti- andpro-apoptotic Bcl-2 proteins is believed to be involved in the“decision” step of apoptosis. Furthermore, interactions among the Bcl-2family of proteins appear to exhibit a defined selectivity andhierarchy. For example, the anti-apoptotic E1B protein showspreferential binding to pro-apoptotic Bcl-2 proteins, whereas thepro-apoptotic Hrk binds only to the anti-apoptotic family member. Thus,the pro-apoptotic protein Bok may regulate apoptosis through similarmechanisms by forming hetero-dimers with selective anti-apoptoticproteins.

[0111] Analysis of the relatedness of amino acid sequences of differentBcl-2 proteins indicated that Bok is not closely related to anyparticular Bcl-2 member and probably diverged early during evolution.The less conserved BH1 domain of Bok may determine its uniquehetero-dimerization property. In both yeast and mammalian cells, Bokinteracts with some but not other anti-apoptotic proteins, suggestingthe possible evolution of selective pairs of death agonists andantagonists with restricted hetero-dimerization properties to confertissue specificity of the death program. Apoptosis induced by Bok intransfected CHO cells could be mediated through inhibition of theprotection afforded by Mcl-1 or other Bok partners. It is likely thatBok may interact with its dimerization partner(s) including Mcl-1 andBfl-1 in reproductive tissues to regulate apoptosis. Of interest, ourrecent data indicated that Mcl-1, but not Bcl-2, is highly expressed inovarian cells. Based on the suppression of Bok-induced apoptosis byBHRF1, it is possible that reproductive tissues expressing Bok arepotential targets for this anti-apoptotic protein encoded by theEpstein-Barr virus. Recent studies have suggested that anti-apoptoticproteins may bind to ced-4/Apaf-1 homologs, which, in turn, activatedownstream caspases. Elucidation of the hetero-dimerization partner(s)for Bok in gonads and uterus would allow characterization of theputative ced-4 homologs in these tissues.

[0112] Recent crystallographic analyses of complexes formed between theanti-apoptotic protein Bcl-xL and the BH3 domain of the pro-apoptoticBak indicated that the α-helix in the BH3 domain of different Bcl-2proteins plays a central role in defining the binding specificity toBcl-xL. Because Bok does not interact with Bcl-xL in the yeasttwo-hybrid system, further studies on the BH3 region of Bok and relatedproteins could define the specificity of hetero-dimerization amongdifferent pro- and anti-apoptotic protein pairs and their role inapoptosis regulation.

[0113] Although over-expression of Bax and Bak induces yeast cell death,the present Bok fusion protein did not affect yeast cell survival. Inaddition, lack of interactions between Bok and different pro-apoptoticBcl-2 proteins in the two-hybrid assay are not due to detrimentaleffects of these death agonists on yeast cells because co-transformationof these apoptosis agonists with Bcl-xL led to activation of thereporter genes. It is likely that moderate expression of these deathagonists using the present expression vector may not significantlyaffect yeast cell survival, thus allowing studies on interactionsbetween different Bcl-2 proteins.

[0114] The majority of ovarian follicles and about 50% of testiculargerm cells undergo apoptosis under normal physiological conditions,whereas the menstruation involves monthly apoptosis of uterineendometrial cells. The restricted expression of Bok in the gonads anduterus suggests its potential role in the regulation of apoptosis inthese tissues. It is likely that selective pairs of Bcl-2agonists/antagonists may play tissue-specific roles in the regulation ofapoptosis. Indeed, mutant mice deficient in Bcl-2 or Bax showedabnormality in apoptosis regulation only in distinct cell lineage.Although Bax-deficient mice showed an accumulation of granulosa cells inatretic follicles, these cells were still apoptotic, suggesting theinvolvement of additional pro-apoptotic factors during ovarian follicleatresia. Because the pro-apoptotic protein Bax has been suggested tofunction as a tumor suppressor gene in colon adenocarcinomas and becauseinactivation of Bax in transgenic mice leads to enhanced tumorigenesis,it would also be interesting to investigate changes in Bok functionduring gonadal and uterine tumorigenesis. Because cyclic variations inreproductive hormones are essential in the regulation of apoptosis ingonadal and uterine tissues, future investigations on the hormonalregulation of the Bok and its dimerization partner (s) in thesereproductive tissues would allow the design of novel strategies tomodulate reproductive functions. These studies could also provideunderstanding on the role of Bok in gonadal and uterine diseasesassociated with aberrant regulation of apoptosis.

EXAMPLE 2

[0115] Characterization of a Bok Splicing Variant with a Truncated BH3Domain, which Induces Apoptosis but Does Not Dimerize withAnti-Apoptotic Bcl-2 Proteins

[0116] A Bok splicing variant is identified in which the region encodedby exon three is absent, creating a truncated short form (Bok-S) of thefull-length Bok protein (Bok-L). The skipping of exon three maintainsthe original reading frame and retains the BH2 and the C-terminalmembrane anchoring domains; however, parts of the BH3 and BH1 domainswere deleted. Functional analysis indicated that Bok-S is still capableof inducing apoptosis. The truncated Bok has lost its ability toheterodimerize with Mcl-1, BHRF-1 and Bfl-1, suggesting that theproapoptotic activity of this variant is not mediated by its binding toantiapoptotic Bcl-2 proteins.

[0117] Materials and Methods

[0118] Reverse Transcription-PCR of the Bok-S Transcript

[0119] Total RNA from different tissues was isolated from 27-day-oldSprague-Dawley rats using an anion exchange resin chromatographic column(Qiagen, Chatsworth, Calif.) before reverse transcription with oligo(dT) 18 as primer in a reaction containing RNase H-free reversetranscriptase from Moloney murine leukemia virus (Clontech, Palo Alto,Calif.). For PCR amplification of Bok cDNAs, aliquots of DNA equivalentto 0.5 μg total RNA were used in each reaction (50 μl). To minimizecontamination during PCR, control reactions containing a single primeror RNA without reverse transcriptase were routinely performed. All PCRwas carried out under high stringency conditions (94° C., 45 s, 68° C.45 s, 72° C. 4 min) for 30 cycles. Isolation of Bok genomic DNA,Southern blot hybridization and genomic analysis. A genomic DNA fragmentwas isolated from a mouse BAC genomic DNA library (Genome Systems Inc.,St. Louis, Mo.) using the full-length Bok cDNA probe. The Bok genomicfragment was first analyzed by restriction enzyme mapping, followed bysubcloning into the pUC18 vector for dideoxy sequencing analysis of bothDNA strands. Overlapping clones were isolated to define the direction ofindividual clones and to facilitate assignment of intron-exon junctions.For Southern blot hybridization analysis, genomic DNA (10 μg) wasdigested with indicated restriction enzymes, separated byelectrophoresis on a 0.8% Agarose gel, and then transferred onto a Nylonmembrane (Hybond-N, Amersham Corp., Arlington Heights, Ill.).Hybridization was performed in the QuickHyb buffer (Clontech) at 60 Cwith ³²P-labeled cDNA probes. The filters were washed with 0.1×SSC and0.5% SDS at 65 C. before exposure. Generation of mutant constructs.Specific mutations in the BH3 domain of Bok-L were generated by atwo-step PCR mutagenesis method using a Bok cDNA template as previouslydescribed (Hsu et al. (1997) Mol Endocrinol 11: 1858-1867). Theresulting PCR products were evaluated for correct size on a 1% Agarosegel, purified, and subcloned into the EcoR1 site of the pcDNA3 vectorfor mammalian cell expression (Invitrogen, Inc., San Diego, Calif.).Truncated Bax and Bak constructs (Bax-S and Bak-S) with homologousdeletion of the BH3-BH1 region found in Bok-S were also generated usingtwo-step overlapping PCR, and subcloned into the EcoRV site of thepcDNA3 vector for eukaryotic cell expression (Hsu et al. supra.) For theyeast two-hybrid assay, mutant Bok-L cDNAs were subcloned into thepGADGH expression vector. Restriction mapping and dideoxy sequencingconfirmed proper orientation and the authenticity of the inserts.

[0120] Yeast Two-Hybrid Assay

[0121] To study dimerization between different Bcl-2 family proteins andvariants or mutants of Bok, cDNAs for Bok-L, Bok-S or Bok mutants werefused to the activation domain (AD) of GAL4 in a yeast shuttle vectorpGADGH. Complementary DNAs encoding different Bcl-2 proteins were fusedto the GAL4-binding domain (BD) of pGBT9. After transformation of yeastcells, colonies containing different protein pairs were selected onplates lacking tryptophan and leucine. To test for specificprotein-protein interactions, positive transformants were furtherselected for growth in media deficient for tryptophan, leucine andhistidine but containing 5-30 mM 3-aminotriazole to inhibit endogenoushistidine production. A minimum of five independent transformantscontaining each pair of fusion cDNAs was routinely analyzed.

[0122] Analysis of Apoptosis in Transfected CHO Cells

[0123] Apoptosis was monitored following transfection of different cDNAsas previously described ( Hsu et al. (1997) Proc. Natl. Acad. Sci. USA94: 12401 CHO cells (2×10⁵ cells/well) were cultured in Dulbecco'smodified Eagle's medium (DMEM)/F12 supplemented with 10% fetal bovineserum, 100 U/ml penicillin, 100 g/ml streptomycin and 2 mM glutamine.One day later, cells were transfected using the lipofectamine procedure(Life Technologies, Gaithersburg, Md.) with the pcDNA3 expression vectorwith or without different cDNA inserts, together with {fraction (1/10)}to {fraction (1/20)} amounts of an indicator plasmid pCMV-β-gal to allowthe identification of transfected cells. Inclusion of a 10-fold excessof expression vectors as compared with the pCMV-β-gal reporter plasmidensured that most of the β-galactosidase-expressing cells also expressedthe protein(s) under investigation. Cells were incubated with plasmidsin a serum-free medium for 4 h, followed by the addition of fetal bovineserum to a final concentration of 5% and further incubation for 1 4 h.After an additional culture in fresh medium for 18 h, cells were fixedby using 0.25% glutaraldehyde and stained with X-gal to detectβ-galactosidase expression. The number of blue cells was counted bymicroscopic examination. Data are expressed as the percentage (mean+/−SEM) of viable cells as compared to the control group. In vitrodirect protein-binding assay. To further demonstrate the specificity ofinteractions between Bok variants and antiapoptotic proteins, directprotein-protein interactions were studied using recombinant Bok andFLAG-tagged BHRF-1 translated in vitro. ³⁵S-methionine labeled ornonlabeled proteins were generated using the TNT coupled reticulocytelysate system (Promega, Madison, Wis.). Pairs of proteins were incubatedin the binding buffer (PBS, 0.2% NP-40 and protease inhibitor cocktail;Sigma, St. Louis, Mo.) for 2 h at 4° C. followed by incubation with 1.5μg of M2 antibody against the FLAG tag (Kodak, Rochester, N.Y.) undergentle agitation. The complexes formed between the antibody andrecombinant proteins were precipitated with Protein A Sepharose(Pharmacia Biotech, Uppsala, Sweden) and resolved using 12-15% SDS PAGE.Following fixation, gels were treated with Amplify fluorographic agents(Amersham Life Science Inc., England) before exposure to x-ray films.

[0124] Statistical Analyses and Sequence Analysis

[0125] One-way analysis of variance followed by Scheffe's F-test wasused to determine the statistical significance of cell viabilityemploying the STATVIEW software (Abacus Concepts, Inc., Berkeley,Calif.). The hydropathicity of protein sequence was analyzed usingBiology Workbench version 2.0 (http://biology.ncsa.uiuc.edu/BW/BW.cgi).

[0126] Results

[0127] Existence of Long and Short Bok Splicing Variants in ReproductiveTissues

[0128] We amplified Bok cDNA from a rat ovarian cDNA library usingprimers flanking the open reading frame (ORF) of Bok. A 513 bp PCRproduct was obtained in addition to the predicted 642 bp band. DNAsequencing of the lower molecular weight product indicated it wasidentical to that of the Bok cDNA except that nucleotides encoding aminoacid 76-118 were missing. This short transcript (Bok-S) encoded a 170amino acid polypeptide and the deletion of 43 amino acids from thefull-length 213 amino acid protein (Bok-L) led to the fusion of theN-terminal half of the BH3 domain to the C-terminal half of the BH1domain. To confirm the authenticity of this variant, reversetranscription-PCR was performed using total RNA from different tissues.Electrophoresis analysis confirmed the presence of a PCR product of 642bp in the ovary, uterus and testis, tissues known to express the Boktranscript. In addition, a lower band of 513 bp was found in the ovary,less in the uterus and absent from the testis. Negative controlreactions using only a single primer or RNA without reversetranscription did not generate any products. Subsequent subcloning andsequencing confirmed that the 642 bp and 513 bp bands encode theexpected Bok-L and Bok-S transcripts, respectively.

[0129] The deduced amino acid sequence of Bok-S showed that apresumptive alternative splicing led to the disruption of both BH1 andBH3 domains of Bok-L, changing the original BH3 sequence [SEQ ID NO:9]71 LLRLGDELEQIR 82 to [SEQ ID NO:10] 71 LLRLGITWGKW 82. However,Kyte-Doolittle hydropathicity analysis suggested that the hydropathicityprofile of the BH3/BH1 fusion region found in Bok-S did not differsubstantially from that of the original BH1 domain in Bok-L.Furthermore, the 5 and 6 regions predicted, based on their homology tosimilar regions in Bax and Bak, were unaltered in the truncated Bok-S.These regions have been postulated to be important for channel formationin the mitochondria by different Bcl-2 proteins.

[0130] Bok Gene Arrangement and the Derivation of Alternative SplicingVariants

[0131] To elucidate the mechanism by which two Bok isoforms weregenerated, the Bok gene and its exon/intron junctions were analyzed.Following the screening of a bacterial artificial chromosome-based mousegenomic DNA library using a mouse Bok cDNA fragment, one genomic clonefor Bok was isolated. The amino acid sequence of the coding region forthe mouse clone was found to be identical to its rat counterpart.Southern blot hybridization of mouse genomic DNA digested with differentrestriction enzymes using cDNA probes corresponding to two differentregions of the Bok gene demonstrated the presence of a single Bok genein the mouse. Further characterization of the genomic clone indicatedthat the entire Bok gene spanned 11 kb and consisted of 5 exons. The BokORF was encoded by sequences in exons II to V whereas exon I containedonly untranslated sequences. Comparison of the ORF of Bok-S with genomicsequences indicated that Bok-S was derived following the splicing out ofexon III.

[0132] Bok-S Promotes Cell Death in Transfected Cells

[0133] To study the role of Bok-S in apoptosis regulation, expressionvectors containing Bok-S in either sense or antisense orientation wereconstructed. Transfection of CHO cells with either Bok-S or Bok-L, butnot the reverse construct, significantly reduced the number oftransfected cells, demonstrating that Bok-S retained its ability toinduce apoptosis despite the loss of the BH3 sequence. In addition, cellkilling induced by either Bok-S or Bok-L was antagonized bycotransfection with P35, a baculoviral-derived caspase inhibitor. Bok-Sdoes not heterodimerize with antiapoptotic Bcl-2 proteins. Because Bokwas isolated based on its ability to bind Mcl-1 and the dimerizationbetween pro- and antiapoptotic Bcl-2 proteins has been suggested to beimportant in apoptosis, we analyzed whether Bok-S that maintained itscell killing ability could still dimerize with antiapoptotic Bcl-2proteins. In the two-hyprid assay, interactions between Bok-S anddifferent Bcl-2 family members were tested. Bok-S did not interact withany Bcl-2 proteins tested whereas Bok-L interacted strongly with Mcl-1,Bfl-1 and BHRF-1, as previously reported. To further confirm findings inyeasts, a direct protein-protein interaction assay was performed usingin vitro translated recombinant Bok variants and the antiapoptoticprotein BHRF-1 that exhibited strongest interaction with Bok-L in yeast.BHRF-1 interacted strongly with Bok-L in vitro but showed negligibleinteraction with Bok-S. Thus, heterodimerization of Bok-S withantiapoptotic Bcl-2 proteins is probably not needed for apoptosisinduction.

[0134] BH3 Mutants of Bok Defective in Heterodimerization Still RetainProapoptotic Activity

[0135] Because Bok-S lacking a BH3 domain still retained its cellkilling potential, we hypothesized that the BH3 domain might bedispensable for the proapoptotic activity of Bok-L. Bok-L cDNAs withalanine or glycine substitutions in the BH3 domain were constructed andthe ability of these mutants to promote apoptosis was studied. Themutants included alanine substitutions at the highly conserved glycine75 or glycine 75 plus flanking aspartic acid 76 and glutamic acid 77(BokADE: G 75 A and BokAAA: 75 AAA 77). In addition, a glycinesubstitution was made for leucine 71 to leucine 74 (BokGGGG: 71 LLRL 74to 71 GGGG 74). Transfection of these Bok-L mutants reduced the numberof viable CHO cells as compared to the group with cells transfected withthe pcDNA3 vector without an insert. In contrast, constructs with mutantcDNAs in reverse orientation had no effect on cell survival. These datasuggested that the BH3 domain in Bok-L is dispensable for apoptosisinduction. We further tested the ability of these BH3 domain mutants ofBok-L to dimerize with antiapoptotic Bcl-2 proteins in the yeasttwo-hybrid assay. Substitution of residues in the BH3 domain of Bok-Labolished its interaction with Mcl-1 or Bfl-1. In addition, the abilityof Bok-L to interact with BHRF-1 was also abated by glycine substitutionat residues 71-74 of Bok-L. Similar to findings using the two-hybridassay, in vitro translated BokGGGG mutant also lost its ability tointeract with Bok-L effectively in the direct protein-proteininteraction test. These data suggested that the cell killing ability ofthese BH3 mutants is not correlated to their ability to dimerize withantiapoptotic Bclproteins.

[0136] Mutants of Bax and Bak with Deletion of Their BH3 DomainResembling Bok-S also Retain Proapoptotic Activity

[0137] Because Bok-L is similar in structure to two other proapoptoticproteins Bax and Bak, deletion mutants with truncation of the BH3-BH1region similar to that found in Bok-S were constructed for theseproteins and named as Bax-S and Bak-S (FIG. 3). Full-length Bak and Bax,like Bok-L and Bok-S, effectively reduced cell viability in the CHO celltransfection assay. Of interest, overexpression of Bax-S or Bak-S alsosignificantly reduced the viability of transfected cells, suggestingthat the BH3-BH1 regions deleted in these two proapoptotic proteins arenot essential for apoptosis induction.

[0138] Discussion

[0139] A naturally occurring variant of Bok with proapoptotic activitybut exhibiting negligible dimerization with antiapoptotic Bcl-2 membersis identified. Bok-S with a 43-amino acid deletion between the BH3 andBH1 domains was likely the result of alternative mRNA splicing, leadingto the skipping of exon three during post-transcriptional modification.Analysis of Bok variants and Bok mutants with alterations in the BH3domain indicated that the BH3 domain of Bok-L is critical forheterodimerization but dispensable for apoptosis induction. Likewise,similar deletions between BH3 and BH1 domains of the homologousproapoptotic proteins Bax and Bak also retained cell killing ability.Thus, Bok-L could promote apoptosis independent of heterodimerizationand Bok-S represents a novel proapoptotic Bcl-2 member capable ofinducing cell death without binding to or interference by antiapoptoticBcl-2 partners. This functional Bok variant with retention of the regionspanning BH1 and BH2 domains and the TM sequence provides a unique modelfor further studies of apoptosis mechanisms regulated by Bcl-2 familyproteins.

[0140] The bifunctional antiapoptotic Bcl-2 proteins play a pivotal rolein the decision step of apoptosis. These proteins, represented byBcl-xL, maintain a channel structure important in the control ofmitochondrial membrane potential and volume homeostasis. Regulation ofthese channels controls the release of cytochrome C essential for theactivation of Apaf-1 and caspases important for apoptosis execution. Theantiapoptotic Bcl-2 proteins also function as docking proteins forproapoptotic Bcl-2 members. Because several mutants of Bcl-2 and Bcl-xLsimultaneously lost antiapoptotic activity and the ability to bindproapoptotic Bcl-2 proteins, it is believed that dimerization ofBclprotein pairs mediated by the consensus BH domains is important inapoptosis regulation. Crystallographic studies and computer modelingshowed that the conserved BH1, BH2 and BH3 domains of Bcl-xL and relatedproteins constitute an elongated hydrophobic cleft capable ofinteraction with the amphipathic helix formed by BH3 domains ofproapoptotic partners. Upon heterodimerization, anti- and proapoptoticBclpartners antagonize the actions of the other. It is likely that oneof the mechanisms by which Bok-L exerts its proapoptotic action isthrough dimerization with antiapoptotic partners.

[0141] Mammalian proapoptotic Bcl-2 proteins can be divided into twosubgroups based on domain arrangement. Together with Bax and Bak, Bok-Lbelongs to the first subgroup showing the conserved BH1, BH2, BH3 and TMdomains. In contrast, members of the second subgroup (BAD, BID, Hrk/DP5,Bik/Nbk and Bim) possess only the BH3 domain, with or without the TMregion. Earlier studies suggested that proapoptotic proteins function byantagonizing the action of antiapoptotic proteins mediated by BH3domains. Mutations in the BH3 domain of proapoptotic proteins abolishedtheir dimerization with antiapoptotic partners and cell killingactivity. In addition, polypeptides containing minimal BH3 domainsequences bind antiapoptotic proteins and induce apoptosis intransfected cells or cell-free systems. More recent studies, however,demonstrated that Bax, like Bcl-xL and Bcl-2, also shows intrinsic ionchannel activity in the artificial membrane. In addition, mutations inthe BH1, 2 or 3 domains of Bax do not affect its ability to promoteapoptosis. Likewise, Bak mutants accelerate chemotherapy-inducedapoptosis independent of its heterodimerization property. These datasuggest that the first subgroup of proapoptotic proteins, including Bax,Bak and Bok, could induce apoptosis through channel formation inaddition to their role as ligands for antiapoptotic Bcl-2 proteins.Because the second BH3-only subgroup members lack the region spanningBH1 and BH2 domains important for pore formation and mainly reside inthe cytoplasm, they are believed to serve as ligands or facilitators ofthe pore forming Bcl-2 proteins.

[0142] Our findings that substitution of conserved residues in the BH3domain of Bok-L abates its ability to dimerize with antiapoptoticproteins are in accord with studies on the BH3 domain of itsproapoptotic homologues. Similarly, truncation of the conserved BH3domain in the naturally-occurring Bok-S variant also disruptedheterodimerization but retained cell killing ability, indicating the BH3domain is dispensable for apoptosis induction. Thus, Bok-S represents anew form of proapoptotic protein consisting of only minimal functionalmodules and manifesting proapoptotic action without direct interactionswith antiapoptotic proteins. As shown in the above data, truncation ofthe region between BH3 and BH1 from Bok-L does not affect the homologousα5 and α6 regions proposed to be important for channel formation in Bax.In addition, the hydropathicity property between the 5″-end of the BH1region and the C-terminal TM domain is not altered by the truncationfound in Bok-S. It is likely that the BH3/1, BH2 and TM domains found inBok-S comprise a module sufficient for mediating apoptosis through aheterodimerization-independent pathway. Future studies on thechannel-forming property of the naturally-occurring Bok-S and otherchannel-forming Bcl-2 proteins are important for understanding themechanisms of apoptosis. The channel-forming hypothesis is furthersupported by the finding that Bax-S and Bak-S with truncation at theBH3-BH1 regions homologous to that of Bok-S also retain proapoptoticactivity. Recent studies also indicated that, during apoptosis,activated caspases cleave the N-terminal BH4 domain of antiapoptoticproteins Bcl-2 and Bcl-xL to yield truncated molecules resembling theproapoptotic Bax, Bak or Bok in terms of the BH domain arrangement. Ofinterest, deletion of the BH4 domain from these antiapoptotic proteinsconfers proapoptotic activity and mitochondrial release of cytochrome C,presumably mediated through the C-terminal channel-forming region.

[0143] Like Bok, splicing variants have been reported for Bcl-2, Bcl-xand Bax genes. The Bcl-xL gene encodes three different variants, eachwith a distinct function; the long form of Bcl-x (L) exhibitsantiapoptotic activity whereas Bcl-x-short and Bcl-x- are proapoptotic.Also, Bcl-2 variants lacking the TM domain show decreased antiapoptoticactivity. The proapoptotic Bax gene also encodes a number of splicingvariants with unknown function.

[0144] At least three mechanisms could be proposed for the action ofproapoptotic Bcl-2 proteins: 1) The subgroup of proapoptotic proteinswith only the BH3 domain (e.g. the soluble BAD protein) heterodimerizeswith membrane-bound antiapoptotic proteins to regulate apoptosis; 2) Thesubgroup of membrane-bound proapoptotic proteins with BH1, BH2, BH3 andTM domains, represented by Bok-L, heterodimerizes with antiapoptoticproteins (Mcl-1/Bfl-1) or functions as mitochondrial channels toregulate apoptosis; and 3) The unique Bok-S variant does not dimerizewith antiapoptotic proteins but probably forms mitochondrial channels toregulate apoptosis. Because Bok-S does not interact with antiapoptoticproteins, apoptosis mediated through Bok-S may be important insituations when unwanted cells need to be eliminated quickly despite thepresence of antiapoptotic proteins in the same cell. In the ovary anduterus known to express high levels of Bok transcripts, Bok-S expressioncould provide a short circuit to promote cell demise inhormone-dependent cell populations that express abundant antiapoptoticproteins (such as Mcl-1) but have to be removed swiftly due to cycliccell turnover during reproductive cycles. The search for novel deathpromoters that interact specifically with proapoptotic Bcl-2 proteinscould also be simplified based on the lack of interaction between Bok-Sand other Bcl-2 proteins. Further characterization of this uniqueproapoptotic protein would allow a better understanding of intracellular mechanism of apoptosis, particularly for hormone-regulated celldeath.

1]. An isolated nucleic acid encoding a mammalian Bok protein. 2]. Theisolated nucleic acid according to claim 1, wherein said Bok proteincomprises the amino acid sequence as set forth in SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6 or SEQ ID NO:8. 3]. The isolated nucleic acidaccording to claim 1, wherein said Bok protein is a BH3^(i) variantprotein. 4]. An isolated nucleic acid comprising at least 18 contiguousnucleotides of the sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 orSEQ ID NO:7. 5]. An isolated nucleic acid that hybridizes understringent conditions to the nucleic acid sequence of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5 or SEQ ID NO:7. 6]. An isolated nucleic acid encodinga BH3i variant of a pro-apoptotic Bok related protein. 7]. The isolatednucleic acid of claim 6, wherein said pro-apoptotic Bok related proteinis Bak or Bax. 8]. An expression cassette comprising a transcriptionalinitiation region functional in an expression host and operably linkedto a nucleic acid having a sequence of the isolated nucleic acidaccording to claim
 1. 9]. A cell comprising an expression cassetteaccording to claim 8 as part of an extrachromosomal element orintegrated into the genome of a host cell as a result of introduction ofsaid expression cassette into said host cell, and the cellular progenyof said host cell. 10]. A method for producing pro-apoptotic protein,said method comprising: growing a cell according to claim 9, wherebysaid protein is expressed; and isolating said protein free of otherproteins. 11]. A purified polypeptide composition comprising at least50% of the protein present as a Bok protein or a fragment thereof. 12].A purified polypeptide composition comprising at least 50% of theprotein present as a BH3^(i) variant of a pro-apoptotic Bok relatedprotein. 13]. A monoclonal antibody binding specifically to a Bokprotein. 14]. A non-human transgenic animal model for Bok gene functionwherein said transgenic animal comprises an introduced alteration in aBok gene. 15]. A method of inducing apoptosis in a susceptible cellpopulation, the method comprising: upregulating expression of Bok or aBH3^(i) variant of a pro-apoptotic Bok related protein in said cellpopulation; wherein apoptosis is induced. 16]. The method of claim 15,wherein said susceptible cell population comprises reproductive tissue.17]. The method of claim 15, wherein said upregulating step comprisesinduction of expression of an endogenous Bok gene. 18]. The method ofclaim 15, wherein said upregulating step comprises introduction andexpression of an exogenous Bok coding sequence. 19]. The method of claim15, wherein said upregulating step comprises introduction and expressionof an exogenous coding sequence for a BH3^(i) variant of a pro-apoptoticBok related protein.